Composition of a polypeptide and an amphiphilic compound in an ionic complex and the use thereof
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example 1
Analysis of the Activity of Different Hedgehog Formulations in a Cell Test
Induction of Alkaline Phosphatase
5000 cells of the murine mesenchymal plutipotent line C3H10T1 / 2 (ATCC CCL-226) are sown in each well of a 96-well microtiter plate. The cells are in DMEM, 2 mM glutamine, 100 IU penicillin / ml, 100 μg streptomycin / ml and 10% foetal calf serum. On the next day the medium is replaced by medium which contains human shh (0, 5 or 50 μg / ml) in different formulations (0, 0.00016, 0.00052, 0.0013, 0.0019 or 0.01% sodium deoxycholate), or the various hedgehog formulations are added directly. The test is stopped after 5 days. For this the supernatants are decanted and the cells are washed once with PBS. The cells are lysed in 50 μl 0.1% Triton® X-100 and frozen at −20° C. After thawing, 25 μl aliquots are used for protein determination and to determine the activity of alkaline phosphatase.
Protein Determination According to the Instructions of the Manufacturer Pierce:
75 μl redistilled H2O ...
example 2
Hydrophobic Ion Pair Titration of hshh (Dimer)
Recombinant human sonic hedgehog protein (dimer, 0.8 mg / ml in 50 mM Tris-Cl, pH 7.4 or in 0.1% Tween 80, 50 mM Tris-Cl, pH 7.4) is admixed with increasing concentrations of sodium deoxycholate. The absorbance at 360 nm is measured as an indicator for turbidity (formation of water-insoluble aggregates composed of ionic protein-detergent complexes). It is clear from FIG. 2 that the transition to water-insoluble aggregates occurs above ca. 0.04% Na deoxycholate. The formation of water-insoluble aggregates can be largely prevented in the presence of 0.1% Tween 80. The stated absorbances are not corrected for dilution.
example 3
Analysis of NGF formulations in a bioactivity assay: Dorsal root ganglion neuron development assay
NGF bioactivity was determined by morphometric analysis of dorsal root ganglion (DRG) neurons developing in vitro. Briefly, lumbar DRG's were dissected from E7-E8 embryonic chickens, freed from surrounding connective tissue and dissociated by triturgation through a fire polished pasteur pipette following digestion with 0.1% trypsin for 20 min at 37° C. Contaminating cells, such as fibroblasts were removed by preplating the entire cell preparation onto plastic tissue culture dishes for 2 h. Under these conditions neurons do not attach to the substrate, while fibroblasts and other non-neuronal cells adhere to the tissue culture plastic. “Clean” neurons were harvested by collecting the supernatant and plated onto poly-omithine / laminin coated plastic dishes (48 wells) at a density of 10,000 cells / well in HAM's F14 medium containing 5% FBS. A dose-response curve for NGF was titrated from app...
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